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| United States Patent |
6,066,449
|
|
Ditkoff
, et al.
|
May 23, 2000
|
Method of detecting metastatic thyroid cancer
Abstract
This invention provides a method of detecting metastatic thyroid cancer in
a subject which comprises detecting circulating thyroid cells in a bodily
fluid sample of the subject by obtaining an appropriate nucleic acid
sample from the bodily fluid sample of the subject; and determining
whether the nucleic acid sample contains a marker sequence. Specifically,
this invention provides wherein the marker sequence is mRNA corresponding
to the reverse transcript of DNA encoding thyroglobulin. Also, this
invention provides wherein the marker sequence is mRNA corresponding to
the reverse transcript of DNA encodes thyroid peroxidase. This invention
further provides a test kit for performing the above-described method.
| Inventors:
|
Ditkoff; Beth Ann (New York, NY);
Chabot; John A. (Rye, NY);
Feind; Carl R. (Alpine, NJ);
LoGerfo; Paul (Grandview, NY)
|
| Assignee:
|
The Trustees of Columbia University in the City of New York (New York, NY)
|
| Appl. No.:
|
840551 |
| Filed:
|
April 15, 1997 |
| Current U.S. Class: |
435/6; 435/91.2; 536/24.33 |
| Intern'l Class: |
C12Q 001/68; C12P 019/34; C07H 021/04; C07H 021/02 |
| Field of Search: |
435/6,91.2
536/24.33
935/77,78
|
References Cited
Other References
Ringel et al., J. Clin. Endocrinol. Metab. 83(12):4189-90, Dec. 1998.
Burchill, S.A., et al. (1994) "Neuroblastoma cell detection by reverse
transcriptase-polymerase chain reaction (RT-PCR) for tyrosine hydroxylase
mRNA." Int. J. Cancer 57: 671-675.
Ditkoff, B.A., et al. (1996) "Detection of circulating thyroid cells in
peripheral blood." Surgery 120: 959-965.
Goldblatt, S.A. and Nadel, E.M. (1965) "Cancer cell in the circulating
blood: a critical review II." Acta Cytologica 9: 6-20.
Jiang, H., et al. (1994) "Induction of differentiation in human
promyelocytic HL-60 leukemia cells activates p21, WAF1/CIP1, expression in
the absence of p53." Oncogene 9: 3397-3406.
Johnson, P.W.M., et al. (1995) "The molecular detection of circulating
tumor cells." British Journal of Cancer 72: 268-276.
Katz, A.E., et al. (1994) "Molecular staging of prostate cancer with the
use of an enhanced reverse transcriptase-PCR assay." Urology 43: 765-775.
Lewis, R. (1997) "RT-PCR may ease testing of patients with throid cancer."
Genetic Engineering News vol. 17, No. 6, p. 1.
Lo Gerfo, P., et al. (1970) "Thyroglobulin in benign and malignant thyroid
disease." JAMA 241: 923-925.
Mattano, L.A., et al. (1992) "Sensitive detection of rare circulating
neuroblastoma cells by the reverse transcriptase-polymerase chain
reaction." Cancer Research 52: 4701-4705.
Seiden, M.V., et al. (1994) "Detection of circulating tumor cells in men
wtih localized prostate cancer." Journal of Clinical Oncology 12:
2634-2639.
|
Primary Examiner: Zitomer; Stephanie
Attorney, Agent or Firm: White; John P.
Cooper & Dunham LLP
Claims
What is claimed is:
1. A method of detecting metastatic thyroid cancer in a subject which
comprises detecting circulating thyroid cells in a bodily fluid sample of
the subject by:
(a) obtaining a nucleic acid sample from the bodily fluid sample of the
subject; and
(b) determining whether the nucleic acid sample from step (a) contains a
marker sequence specific for thyroid cells so as to thereby detect
metastatic thyroid cancer in the subject.
2. The method of claim 1, wherein the marker sequence comprises mRNA
encoding thyroglobulin, and wherein the determining of step (b) comprises:
(i) amplifying a reverse transcript of the thyroglobulin encoding mRNA
present in the nucleic acid sample of step (a); and
(ii) detecting the presence of the reverse transcript of the thyroglobulin
encoding mRNA in the resulting amplified nucleic acid.
3. The method of claim 2, wherein the amplification of step (I) is
performed with a pair of primers that are complementary to the reverse
transcript.
4. The method of claim 3, wherein the primers that are complementary to the
reverse transcript comprise a first primer complementary to exon 1 of the
nucleic acid encoding thyroglobulin and a second primer complementary to
exon 5 of the nucleic acid encoding thyroglobulin.
5. The method of claim 4, wherein the first primer is
5'-GCCTCCATCTGCTGGGTGTC-3' SEQ ID NO: 1.
6. The method of claim 4, wherein the second primer is
5'-CTCCCTCCGCAGAACACTGGGGT-3' SEQ ID NO: 2.
7. The method of claim 1, wherein the marker sequence comprises mRNA
encoding thyroid peroxidase, and wherein the determining of step (b)
comprises:
(i) amplifying a reverse transcript of thyroid Peroxidase encoding mRNA
present in the nucleic acid sample of step (a); and
(ii) detecting the presence of the reverse transcript of the thyroid
peroxidase encoding mRNA in the resulting amplified nucleic acid.
8. The method of claim 7, wherein the amplification of step (I) is
performed with a pair of primers.
9. The method of claim 8, wherein one primer of the pair of primers is
5'-AGGAGTCTCGTGTCTCTAG-3' SEQ ID NO: 4.
10. The method of claim 8, wherein one primer of the pair of primer is
5'-GACTGAAGCCGTCCTCATA-3' SEQ ID NO: 5.
11. The method of claim 1, wherein the bodily fluid sample comprises blood.
12. The method of claim 1, wherein the subject is human.
13. The method of claim 1, wherein the nucleic acid sample is obtained at
least three weeks after removal of the thyroid cancer from the subject.
14. A test kit for detecting circulating metastatic thyroid cancer in a
subject which comprises a means for detecting a marker sequence, wherein
the marker sequence is specific for said thyroid cells and said means for
detection is selected from the group consisting of:
a) a pair of primers, wherein one primer is 5'-GCCTCCATCTGCTGGGTGTC-3' (SEQ
ID NO: 1) and another primer is 5'-CTCCCTCCGCAGAACACTGGGGT-3' (SEQ ID NO:
2) and;
b) a pair of primers, wherein one primer is 5'-AGGAGTCTCGTGTCTCTA G-3' (SEQ
ID NO: 4) and another primer is 5'-GACTGAAGCCGTCCTCATA-3' (SEQ ID NO: 5)
wherein detection of the marker sequence thereby indicating metastatic
thyroid cancer in the subject.
Description
Throughout this application, various publications are referenced by author
and date. Full citations for these publications may be found listed
alphabetically at the end of the specification immediately before the
claims. The disclosures of these publications in their entireties are
hereby incorporated by reference into this application in order to more
fully describe the state of the art as known to those skilled therein.
BACKGROUND OF THE INVENTION
Differentiated thyroid cancer is the most common endocrine malignancy
(Cady, B. and Rossi, R. L. , 1991). In the United States, there are
approximately 14,000 new patients and 1,100 deaths per year (Shah, J. P.
and Lydiatt, W., 1995). Follicular cancers generally metastasize via
hematogenous dissemination, whereas papillary cancers spread through
lymphatic involvement. Patients with distant metastatic disease have the
worst prognosis (Braverman, L. E., et al., 1991).
Current techniques to detect metastases include nuclear scans (such as
.sup.131 I scanning) as well as measurement of serum thyroglobulin.
Detectable serum thyroglobulin levels are found in normal patients as well
as some types of benign thyroid disease (LoGerfo, P., et al., 1979).
Thyroglobulin is a large glycoprotein (molecular weight of 660,000)
secreted exclusively by the thyroid follicular cell and is often elevated
in patients with differentiated thyroid malignancies.
Thyroid peroxidase is a membrane-bound glycosylated, hemoprotein enzyme
that plays a key role in thyroid hormone biosynthesis by catalyzing both
the iodination of tyrosyl residues and the coupling of iodotyrosyl
residues in thyroglobulin. Until 1985, this was considered to be its only
role in the thyroid; however, thyroid peroxidase is closely related to, if
not identified with, the thyroid microsomal antigen associated with the
antithyroid microsomal autoantibodies found in the serum of many patients
with autoimmune thyroid disease.
The detection of small numbers of circulating thyroid cells has previously
been impossible due to insensitive techniques. With polymerase chain
reaction (PCR), however, it is now possible to utilize a tissue-specific
gene expression approach to detect circulating thyrocytes. This technique
has also been utilized with other solid tumors such as prostate and
neuroblastoma (Moreno, J. G., et al., 1992; Katz, A. E., et al., 1994;
Seiden, M. V., et al., 1994; Mattano, L. A., et al., 1992; Burchill, S.
A., et al., 1994; Johnson, P. W. M., et al., 1995). Thyroglobulin and
thyroid peroxidase are two examples of proteins that are expressed
specifically in thyrocytes. Since it is believed that patients with
thyroid cancer and metastatic thyroid cancer will have circulating thyroid
cells in the peripheral bloodstream, whereas those patients without
thyroid cancer will not, one could use PCR to amplify the mRNA transcripts
of these proteins in order to detect circulating thyroctyes. The detection
of circulating thyrocytes may have important significance in the diagnosis
and prognosis of thyroid cancer.
SUMMARY OF THE INVENTION
This invention provides a method of detecting metastatic thyroid cancer in
a subject which comprises detecting circulating thyroid cells in a bodily
fluid sample of the subject by obtaining an appropriate nucleic acid
sample from the bodily fluid sample of the subject; and determining
whether the nucleic acid sample contains a marker sequence.
In a specific embodiment of the above-described method, the marker sequence
is mRNA corresponding to the reverse transcript of DNA encoding
thyroglobulin. One skiled in the art may determine whether the nucleic
acid sample contains mRNA corresponding to the reverse transcript of DNA
encoding thyroglobulin by amplifying the nucleic acid present in the
nucleic acid sample, and detecting the presence of thyroglobulin in the
resulting amplified nucleic acid. One could amplify the nucleic acid using
a pair of appropriate primers.
In another embodiment of the above-described method, the marker sequence is
mRNA corresponding to the reverse transcript of DNA encodes thyroid
peroxidase.
This invention also provides when bodily fluid sample of the
above-described method comprises blood.
This invention also provides when the subject of the above-described method
is human.
This invention also provides a test kit for performing the above-described
method. Specifically, this invention also provides the above-described
test kit, wherein the kit comprises specific primers specific for
amplification of nucleic acid encoding thyroglobulin or thyroid
peroxidase.
BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A and 1B
1A. Sequences of the primers used to detect thyroglobulin. Primer P1 (Seq.
ID No. 1) and primer P2 (Seq. ID No. 2).
1B. Schematic representation of a portion of the thyroglobulin gene showing
the relative position of the specific PCR primers. Primers P1 and P2 bind
sequences such that RT-PCR amplification yields an approximately 529
base-pair transcript (FIG. 2A). Primer P1 to Primer P2 genomic DNA length
measures approximately 6.5 kbp.
FIGS. 2A and 2B
2A. Thyroglobulin reverse transcriptase polymerase chain reaction (RT-PCR).
RT-PCR assay identifies expression of thyroglobulin (approximately 529 bp
band) in the peripheral blood of patients with metastatic thyroid cancer
and in a specimen of human thyroid tissue. Inability to detect
thyroglobulin synthesizing cells in peripheral blood of patients with
benign thyroid disease or healthy control volunteers is demonstrated by
the absence of the 529 bp band.
2B. Human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reverse
transcriptase polymerase chain reaction (RT-PCR). GAPDH primers were
utilized in a parallel reaction and these primers amplified the
appropriate 711 bp fragment from all specimens.
FIG. 3 Verification of amplified fragment by restriction enzyme mapping.
Amplified thyroglobulin fragments were isolated from agarose gels by
electroelution and DNA was digested with restriction enzymes and analyzed
on an agarose gel. The uncut sample (U) measures approximately 529 bp,
while the cut samples (C) are approximately 257 bp and 272 bp,
respectively. Paired samples (both uncut and cut) are shown for a
representative nine patients.
FIG. 4 Nucleotide sequence of thyroglobulin RT-PCR fragment SEQ ID No. 3.
FIGS. 5A and 5B
5A. Sensitivity of reverse transcriptase polymerase chain reaction (RT-PCR)
assay determined by analysis of diluted human follicular thyroid cells.
RNA was extracted from serial dilutions of human follicular thyroid cells
in 5 cc whole blood. A single band of approximately 529 bp was identified
when 10.sup.3 cells per mL were analyzed. No band was identified in
unspiked blood.
5B. As above except with thyroid stimulating hormone (TSH) stimulation. A
single band of approximately 529 bp was identified when 10.sup.2 cells per
mL were analyzed. No band was identified in unspiked blood.
FIGS. 6A and 6B
6A. RT-PCR assay identifies expression of thyroglobulin (approximately 529
bp band) in the peripheral blood of thyroid cancer patients. Samples were
obtained from thyroid cancer patients immediately before (B) and after (A)
thyroid surgery, and in a specimen of human thyroglobulin (+) in patient
#1 this band was identified in both the pre- and postoperative blood
samples. However, in patient #2, this band was only identified during the
immediate postoperative period. The inability to detect thyroglobulin
synthesizing cells in the preoperative peripheral blood samples of patient
#2 is demonstrated by the absence of the 529 bp band.
6B. Human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reverse
transcriptase polymerase chain reaction (RT-PCR). GAPDH primers were
utilized in a parallel reaction and these primers amplified the
appropriate 711 bp fragment from all specimens.
FIGS. 7A, 7B, 7C and 7D
7A. Sequence of primer used to detect thyroid peroxidase. Primer P1 (Seq.
ID No. 4).
7B. Sequence of primer used to detect thyroid peroxidase. Primer P2 (Seq.
ID No. 5).
7C. Sequence of thyroid peroxidase RT-PCR fragment using primer P1 (Seq. ID
No. 6).
7D. Sequence of thyroid peroxidase RT-PCR fragment using primer P2 (Seq. ID
No. 7).
DETAILED DESCRIPTION OF THE INVENTION
This invention provides a method of detecting metastatic thyroid cancer in
a subject which comprises detecting circulating thyroid cells in a bodily
fluid sample of the subject by obtaining an appropriate nucleic acid
sample from the bodily fluid sample of the subject; and determining
whether the nucleic acid sample contains a marker sequence.
As described herein, "marker sequence" means a sequence that encodes a
protein that is specifically expressed in only thyrocytes. Thyroglobulin
and thyroid peroxidase are two examples of protein that are expressed in
only thyroid cells. One skilled in art could determine other proteins that
are expressed soley in thyroid cells using well-known methods and
techniques. After such determination, one could determine the sequence of
such protein using known methods in the art, e.g. Sanger's dideoxy method.
As describe herein, "bodily fluid sample" includes any fluid sample from
the body of the subject that one can obtain a nucleic acid sample so as to
determine whether the nucleic acid sample contains the marker sequence.
One such example is blood, specifically peripheral blood. However, one
skilled in the art could obtain other bodily fluid samples, such as
bronchial fluids, that would be able to determine whether the sampel
contains a marker sequence so as to determine whether circulating
thyrocytes exist.
One skilled in the art could be able to practice the subject invention in
various subjects, e.g. animals, but specifically humans.
In a specific embodiment of the above-described method, this invention
provides wherein the marker sequence comprises mRNA corresponding to the
reverse transcript of DNA encoding thyroglobulin. One detects the marker
sequence by amplifying the nucleic acid present in the nucleic acid sample
and detecting the presence of thyroglobulin in the resulting amplified
nucleic acid.
One can amplify the nucleic acid by using a pair of appropriate primers.
An example of appropriate primers comprise a first primer complementary to
exon 1 of the nucleic acid molecule encoding thyroglobulin and a second
primer complementary to exon 5 of the nucleic acid molecule encoding
thyroglobulin. Specifically, one primer is 5'-GCCTCCATCTGCTGGGTGTC-3' SEQ
ID NO: 1 and another primer is 5'-CTCCCTCCGCAGAACACTGGGGT-3' SEQ ID NO: 2.
As used herein, "appropriate primer" means any nucleic acid molecule,
naturally or synthetically available, that is able to amplify the nucleic
acids that correspond to the marker sequence in the nucleic acid sample so
as detect the marker sequence. For example, based on the known technology
of polymerase chain reaction, one could design primers appropriate for use
in the above-described method using well-known methods in the art, e.g.
DNA synthesizer.
In a specific embodiment of the above-described method, this invention
provides wherein the marker sequence comprises mRNA corresponding to the
reverse transcript of DNA encodes thyroid peroxidase. One can detect the
marker sequence by amplifying the nucleic acid present in the nucleic acid
and detecting the presence of thyroid peroxidase in the resulting
amplified nucleic acid.
One can amplify the above-described method by using two primers.
Specifically, one primer is 5'-AGGAGTCTCGTGTCTCTAG-3' SEQ ID NO: 4 and
another primer is 5'-GACTGAAGCCGTCCTCATA-3' SEQ ID NO: 5.
This invention further provides a test kit for performing the
above-described method.
In a specific embodiment of the test kit, the kit comprises a pair of
primers, such that one primer is 5'-GCCTCCATCTGCTGGGTGTC-3' SEQ ID NO: 1
and another primer is 5'-CTCCCTCCGCAGAACACTGGGGT-3' SEQ ID NO: 2.
In another specific embodient, the kit comprises a pair of primers, such
that one primer is 5'-GCCTCCATCTGCTGGGTGTC-3' SEQ ID NO: 1 and the other
primer is 5'-GACTGAAGCCGTCCTCATA-3' SEQ ID NO: 5.
This invention is illustrated in the Experimental Details section which
follows. This section is set forth to aid in an understanding of the
invention but is not intended to, and should not be construed to, limit in
any way the invention as set forth in the claims which follow thereafter.
EXPERIMENTAL DETAILS
FIRST SET OF EXPERIMENTS
METHODS
1. Patients
After approval by the human study committee and protocol review boards,
postoperative thyroid cancer patients at Columbia-Presbyterian Medical
Center were invited to participate in this project. Postoperative
peripheral blood samples from 100 patients, including patients with known
metastatic thyroid cancer (6 papillary and 3 follicular cancers) and
patients with thyroid cancer and no evidence of current disease (63
papillary, 10 follicular and 5 patients with both papillary and follicular
cancers). In addition, six postoperative benign thyroid disease (nontoxic
nodular goiters) patients and seven healthy control volunteers were
recruited for the study.
The study includes: 77 females and 23 males with an average age of 53
years. Blood samples were collected from each participant during a routine
follow-up appointment at various time intervals, from one day to 35 years
after thyroid surgery, with an average follow-up of 8.3 years. Patient
information, including history, operative reports, pathology reports,
physical exam and laboratory and radiographic studies, was obtained by
independent chart review. Investigators who performed the RT-PCR assay
were blinded to the patients' clinical characteristics, and the clinical
investigators were blinded to the RT-PCR results. No clinical decisions
were made based on the results of this assay.
Patients were determined to be disease free by a combination of clinical
examination, laboratory and radiographic tests. Operations were
individualized for each patient, ranging from partial to total
thyroidectomies with or without unilateral or bilateral modified or
radical neck dissections. All patients with metastatic disease underwent
at least total thyroidectomies.
2. Blood Preparation for RNA Extraction
Approximately five mL of venous blood was obtained using standard
venipuncture technique, and immediately mixed with 10 mL of RNA STAT 60
(TEL-TEST "B , INC., Friendswood, Tex.) and stored at -20.degree. C. until
RNA extraction.
3. RNA Extraction
Total cellular RNA was extracted from a thyroid follicular tumor cell line
(UCLA R0 82 W-1, a generous gift from G. J. F. Juillard, Los Angeles,
Calif.) (Van Herle, A. J., 1990), thyroid tissue obtained from patients
undergoing thyroidectomy, and whole blood samples from patients and normal
individuals using RNA STAT-60 according to manufacturer's instructions.
The RNA pellet was washed with cold 75% ethanol, dried, dissolved in DEPC
treated water and stored at -80.degree. C. RNA concentrations were
determined by measuring OD at 260 and 280 nm.
4. Oligonucleotide Primers
Two primers (P1 and P2) were custom designed with high specificity to the
thyroglobulin gene (FIG. 1A). All primers were synthesized and gel
purified by Genset (La Jolla, Calif.). These probes bind exons I and V
(FIG. 1B). Primers span a genomic DNA length of approximately 6.5 kb and
bind sequences such that RT-PCR amplification yield an appromixately 529
base-pair transcript (FIG. 2A).
5. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
RT-PCR was carried out as described by Burchill (Burchill, S. A., et al.,
1994). In brief, 1 ug of total RNA was reverse transcribed with 200 units
of Superscript.TM. II (Gibco-BRL, Maryland) in 20 uL of RT stock buffer
containing PCR buffer (200 mM Tris.HCl, pH 8.4, 500 mM KCl), 5 mM
MgCl.sub.2, 500 uM dNTP, 10 U RNase inhibitor (Gibco-BRL), and 100 pmoles
of primer P2. The reaction mixture was covered with 20 uL of mineral oil
and the reverse transcription was allowed to proceed at 38.degree. C. for
2 h . Then 80 uL of PCR master mixture containing PCR buffer (200 mM
Tris.HCl, pH 8.4, 500 mM KCl), 1.25 mM MgCl.sub.2, 100 pmoles primer P1
and P2, and 2.5 units of Taq DNA polymerase (Gibco-BRL, Maryland) was
added. PCR was carried out in a thermocycler (Barnsted/Thermolyne, Iowa)
with an initial denaturation at 94.degree. C. for 5 min., followed by 35
cycles of 94.degree. C. for 1 min., 58.degree. C. for 2 min., 72.degree.
C. for 3 min. Additional extension at 72.degree. C. for 15 min. was
allowed to proceed and then the sample was cooled to 4.degree. C. Twenty
uL of the RT-PCR product was separated in a 2% agarose gel containing
ethidium bromide (0.5 ug/mL) and visualized on a transilluminator. One kb
DNA ladder molecular weight marker (Gibco BRL, Maryland) was used as a
standard.
To confirm the integrity of RNA, RT-PCR was performed with human
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers
5'-CGTCTTCACCACCATGGAGAA-3' SEQ ID NO: 8 and 5'-CATGGCCTCCAAGGAGTAAGA-3'
SEQ ID. NO: 9 (GenBank accession number J02642) as described before
(Jiang, H., et al., 1994) (FIG. 2B).
6. Restriction Enzyme Digestion of RT-PCR Amplified Thyroglobulin Fragment
All RT-PCR amplified thyroglobulin fragments were isolated from agarose
gels by electroelution, ethanol precipitated and DNA pellets were
dissolved in TE (pH 8.0) buffer (Sambrook, J., et al., 1989).
Approximately 1 ug DNA sample was digested with restriction enzyme Bgl II
(Gibco-BRL) at 37.degree. C. for 2 h. and analyzed on 2% agarose gel
containing ethidium bromide (0.5 ug/mL) as above (FIG. 3).
7. Cloning and Sequencing of RT-PCT Amplified Thyroglobulin Fragment
RT-PCR amplified thyroglobulin fragment from human thyroid tissue RNA was
cloned into TA cloning vector pCR II (Invitrogen, California) according to
manufacturer's instructions and sequenced by Sanger's dideoxynucleotide
chain termination method (Sanger, F., et al., 1989)) using SP6 and T7
primers.
8. Thyroglobulin RT-PCR Sensitivity Assay
Thyroglobulin RT-PCR sensitivity was determined by spiking thyroid
follicular carcinoma cells (UCLA R0 82 W-1) (Van Herle, A. J., et al.,
1990) into whole blood samples from normal donors. Ten fold serial
dilutions of thyroid follicular carcinoma cells were mixed with 5 mL
aliquots of whole blood and RNA was isolated as described before. RT-PCR
was performed using 1 ug of total RNA and analyzed as described above
(FIG. 5A). In a second experiment thyroid follicular carcinoma cells were
stimulated in culture with thyroid stimulating hormone (TSH from bovine
pituitary, Sigma Chem. Co., Missouri) at a concentration of 5 uU/mL for 5
days, and the stimulated cells were spiked as above, RNA was isolated and
RT-PCR was performed as before (FIG. 5B).
RESULTS
1. Detection of Thyroglobulin Transcripts by RT-PCR
FIGS. 1A and B show thyroglobulin transcript primers P1 (sense) and P2
(antisense), and their relative positions in the thyroglobulin gene.
Thyroglobulin transcripts were detected as an approximately 529 bp
amplification fragment in human thyroid follicular cell line, human
thyroid tissue and whole blood of metastatic thyroid cancer patients by
reverse transcription of RNA using primer P2 followed by PCR with primers
P1 and P2. FIG. 2A shows examples of thyroglobulin fragment amplification
in thyroid tissue of a patient, and peripheral blood of another patient
with metastatic thyroid cancer. Such an amplification band was not visible
in the peripheral blood of patients with benign thyroid disease and
healthy controls. Intactness of RNA in all cases was confirmed by running
an RT-PCR in parallel with human glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) primers. In each case an amplification band of 711 bp was evident
in the gel (FIG. 2B).
2. Restriction Enzyme Fragmentation and Nucleotide Sequence Verification of
RT-PCR Amplified Fragments
All thyroglobulin RT-PCR fragments were further analyzed by Bgl II
restriction enzyme digestion. FIG. 3 shows a few such analyses. As
expected in each case two restriction fragments of 272 and 257 bp were
revealed confirming the restriction site in the amplified thyroglobulin
fragment. Undigested controls were included in each case in the gel
analysis.
In two representative cases the RT-PCR amplified thyroglobulin fragments
were cloned into pCR II vector (Invitrogen, California) and sequenced in
both directions using SP6 and T7 primers by dideoxynucleotide chain
termination method of Sanger et al (Sanger, F., et al., 1977). The derived
nucleotide sequences of RT-PCR amplified thyroglobulin fragments were
identical to the nucleotide sequence of thyroglobulin reported in the Gen
Bank (Accession number 02154) (Jiang, H., et al., 1994) (FIG. 4). This
further confirmed that the amplified fragments were derived from
thyroglobulin transcripts.
3. Determination of Sensitivity of Thyroglobulin RT-PCR Assay
To determine the sensitivity of this assay initially unstimulated human
thyroid follicular tumor cells (UCLA RO82 W-1) were used for spiking into
peripheral blood of a normal donor. FIG. 5A shows a faint band at 529 bp
when the serial dilution reached to 10.sup.3 tumor cells in 5 mL blood
suggesting the detection limit of 200 tumor cells/mL blood. However, when
the cell spiking experiment was repeated with bovine pituitary thyroid
stimulating hormone (TSH) stimulated thyroid follicular tumor cells, 529
bp RT-PCR band was visible in serial dilution up to 10.sup.2 tumor cells
in 5 mL blood (FIG. 5B). Thus there was a 10 fold increase in sensitivity
with a detection limit of 20 tumor cells/mL blood. All specimens of human
thyroid tissue were found to be positive for thyroglobulin mRNA.
4. Patients
Postoperative peripheral blood samples were obtained from 100 patients,
including patients with known metastatic thyroid cancer (6 papillary and 3
follicular cancers), thyroid cancer and no evidence of current metastases
(63 papillary, 10 follicular and 5 patients with both papillary and
follicular cancers), benign thyroid disease (6 nontoxic nodular goiters)
and normal volunteers (Seiden, M. V., et al., 1994). Thyroglobulin
transcripts were detected in 9/9 patients with metastatic thyroid cancer,
7/78 patients without current metastases, 0/6 patients with benign thyroid
disease and 0/7 normal volunteers (Table 1).
TABLE 1
______________________________________
Comparison of PCR to clinical staging in postoperative patients
RT-PCR
______________________________________
Papiliary thyr 12/69
With current metastates 6/6
Without current
metastates 6/63
Metastates previously 5/22
No history of
1/41
Follicular thyroid 3/13
With current
metastases 3/3
Without current metastases 0/10
Papillary and
1/5
With current metastases 1/5
Without current
metastases 0/0
Benign thyroid 0/6
Control volunteers
0/7
______________________________________
The patients without current metastases (n=78) were further subdivided into
patients who had a history of metastatic disease that had been
successfully treated with either radioactive iodine.sub.-- or surgery
(n=22) and those who never had any metastatic disease (n=56). Of the
patients who had been successfully cured of metastatic thyroid cancer, 5
were positive by PCR analysis. These five patients represent patients who
had papillary thyroid cancer that was metastatic to lymph nodes.
Two additional patients had thyroglobulin transcripts detected in
peripheral blood samples. One patient with a papillary thyroid cancer had
a concurrent parathyroid cancer, and one patient had both papillary and
follicular thyroid cancers. In addition, there were four other patients
with both papillary and follicular thyroid cancers who tested negative by
RT-PCR analysis.
Not all patients underwent serum thyroglobulin tests. However, the range of
serum thyroglobulin levels for patients with documented metastatic disease
ranged from 16 to 46,000 and did not always correlate with extent of
disease. Not all patients were taken off their thyroid hormone prior to
obtaining serum thyroglobulin levels. Four of the seven RT-PCR positive
thyroid cancer patients without current metastatic disease had serum
thyroglobulin levels drawn; all of these tests were normal. In addition,
three of these seven patients underwent total body .sup.131 I scans which
were also negative for metastatic disease.
CONCLUSIONS
Until recently, small numbers of circulating tumor cells could not be
detected in the peripheral blood (Goldblatt, S. A., and Nadel, E. M.,
1965). However, with the advent of PCR, it is now possible to amplify
tumor-specific transcripts by RT-PCR. These techniques have been used to
identify blood-borne tumor cells in several solid cancers including
melanoma, prostate and neuroblastoma (Moreno, J. G., et al., 1992; Katz,
A. E., et al., 1994; Seiden, M. V., et al., 1994; Mattano, L. A., et al.,
1992; Burchill, S. A., et al., 1994; Johnson, P. W. M., et al., 1995). In
prostate cancer especially, the detection of prostate-specific antigen
sequences has been shown to correlate with the extent of disease (Katz, A.
E., et al., 1994).
Using RT-PCR technique, a test for the detection of blood-borne
thyroglobulin synthesizing cells was developed. Because thyroglobulin is
secreted only by thyroid cells, we believe these circulating cells are
thyrocytes. Currently, the limit of sensitivity of this assay using TSH
stimulated follicular thyroid cells is 20 cells/mL of whole blood.
Burchill et al cite a sensitivity of 1-10 cells per mL of whole blood when
utilizing RT-PCR for the detection of neuroblastoma cells (Burchill, S.
A., et al., 1994). Thyrocytes were identified in all patients with
metastatic thyroid cancer, but not in any of the control patients with
benign thyroid disease.
Of the 78 thyroid cancer patients tested who appeared to be clinically
disease free (by physical exam, serum thyroglobulin level and/or .sup.131
I total body scanning), 7 patients were positive by RT-PCR assay. These
patients include 5 patients with papillary thyroid cancer who had been
treated for lymph node metastases in the past, as well as one patient with
papillary thyroid cancer and a concurrent parathyroid cancer and one
patient with both papillary and follicular thyroid cancers. This subset of
RT-PCR positive patients could represent false positives, or they may
signify subclinical disease. On the other hand, the presence of
circulating thyroid cells may be a harbinger of future clinically apparent
metastatic disease. Since thyroid cancer is usually a slow growing
indolent tumor and disease recurrence is possible decades after the
initial treatment, these positive patients may be at risk for the future
development of thyroid cancer metastases.
It is generally recognized that papillary thyroid cancers metastasize to
the lymph nodes, and follicular cancers are hematogenously disseminated
(Braverman, L. E., et al., 1991). Thus it is an unexpected result to find
that all six patients with metastatic papillary thyroid cancer were found
to have thyrocytes circulating in the peripheral bloodstream. In addition,
of the 7 thyroid cancer patients without evidence of disease but with
positive RT-PCR results, all had papillary thyroid cancer. This finding
further supports the hypothesis that papillary cancers may also
metastasize hematogenously. The discovery of circulating thyrocytes in the
peripheral blood of patients with metastatic thyroid cancer may be a key
to understanding the method of tumor dissemination and spread.
SECOND SET OF EXPERIMENTS
METHODS
1. Patients
All patients undergoing thyroid and/or parathyroid operation at
Columbia-Presbyterian Medical Center were invited to participate in this
project. Immediate preoperative and postoperative peripheral blood samples
were obtained from 36 patents undergoing thyroid or parathyroid operation.
These patients included 9 with differentiated thyroid cancer, 19 with
benign thyroid disease, 3 with parathyroid adenomas, 4 with differentiated
thyroid cancer and parathyroid adenomas and one with both benign thyroid
disease and parathyroid adenoma. In addition, 10 of the 36 patients had
blood samples drawn 3 weeks postoperatively.
The experiment includes 31 females and 5 males with an average of 50.3
years. Patient information, including history, operative reports,
pathology reports, physical exam and laboratory and radiographic studies,
was obtained by independent chart review. Investigators who performed the
RT-PCR assay were blinded to the patients' clinical characteristics, and
the clinical investigators were blinded to the RT-PCR results. No clinical
decisions were made based on the results of this assay.
Operations were individualized for each patient, ranging from partial to
total thyroidectomies with or without neck exploration and
parathyroidectomy, depending on the clinical indications. All patients
with thyroid cancer underwent total thyroidectomy.
2. Blood Preparation for RNA Extraction
Approximately five mL of venous blood was obtained using standard
venipuncture technique, and immediately mixed with 10 mL of RNA STAT 60
(TEL-TEST "B , INC., Friendswood, Tex.) and stored at -20.degree. C. until
RNA extraction.
3. RNA Extraction
Total cellular RNA was extracted from a thyroid follicular tumor cell line
(UCLA R0 82 W-1, a generous gift from G. J. F. Juillard, Los Angeles,
Calif.) (Van Herle, A. J., 1990), thyroid tissue obtained from patients
undergoing thyroidectomy, and whole blood samples from patients and normal
individuals using RNA STAT-60 according to manufacturer's instructions.
The RNA pellet was washed with cold 75% ethanol, dried, dissolved in DEPC
treated water and stored at -80.degree. C. RNA concentrations were
determined by measuring OD at 260 and 280 nm.
4. Oligonucleotide Primers
Two primers (P1 AND P2) were custom designed with high specificity to the
thyroglobulin gene (FIG. 1A). All primers were synthesized and gel
purified by Genset (La Jolla, Calif.). These primers bind exons I and V
(FIG. 1B). Primers span a genomic DNA length of approximately 6.5 kb and
bind sequences such that RT-PCR amplification yield an appromixately 529
base-pair transcript.
5. Reverse Transcriptase-polymerase Chain Reaction (RT-PCR)
RT-PCR was carried out as described by Burchill (Burchill, S. A., et al.,
1994). In brief, 1 ug of total RNA was reverse transcribed with 200 units
of Superscript.TM. II (Gibco-BRL, Maryland) in 20 uL of RT stock buffer
containing PCR buffer (200 mM Tris.HCl, pH 8.4, 500 mM KCl), 5 mM
MgCl.sub.2, 500 uM dNTP, 10 U RNase inhibitor (Gibco-BRL), and 100 pmoles
of primer P2. The reaction mixture was covered with 20 uL of mineral oil
and the reverse transcription was allowed to proceed at 38.degree. C. for
2 h . Then 80 uL of PCR master mixture containing PCR buffer (200 mM
Tris.HCl, pH 8.4, 500 mM KCl), 1.25 mM MgCl.sub.2, 100 pmoles primer P1
and P2, and 2.5 units of Taq DNA polymerase (Gibco-BRL, Maryland) was
added. PCR was carried out in a thermocycler (Barnsted/Thermolyne, Iowa)
with an initial denaturation at 94.degree. C. for 5 min., followed by 35
cycles of 94.degree. C. for 1 min., 58.degree. C. for 2 min., 72.degree.
C. for 3 min. Additional extension at 72.degree. C. for 15 min. was
allowed to proceed and then the sample was cooled to 4.degree. C. Twenty
uL of the RT-PCR product was separated in a 1.6% agarose gel containing
ethidium bromide (0.5 ug/mL) and visualized on a transilluminator. One kb
DNA ladder molecular weight marker (Gibco BRL, Maryland) was used as a
standard.
To confirm the integrity of RNA, RT-PCR was performed with human
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) p r i me r s
5'-CGTCTTCACCACCATGGAGAA-3' SEQ ID NO: 8 and 5'-CATGGCCTCCAAGGAGTAAGA-3'
SEQ ID NO: 9 (GenBank accession number J02642) as described previously
(Jiang, H., et al., 1994).
6. Restriction Enzyme Digestion of RT-PCR Amplified Thyroglobulin Fragment
All RT-PCR amplified thyroglobulin fragments were isolated from agarose
gels by electroelution, ethanol precipitated and DNA pellets were
dissolved in TE (pH 8.0) buffer (Sambrook, J., et al., 1989) .
Approximately 1 ug DNA sample was digested with restriction enzyme Bgl II
(Gibco-BRL) at 37.degree. C. for 2 h. and analyzed on 1.6% agarose gel
containing ethidium bromide (0.5 ug/mL) as above.
7. Cloning and Sequencing of RT-PCT Amplified Thyroglobulin Fragment
RT-PCR amplified thyroglobulin fragment from human thyroid tissue RNA was
cloned into TA cloning vector pCR II (Invitrogen, California) according to
manufacturer's instructions and sequenced by Sanger's dideoxynucleotide
chain termination method (Sanger, F., et al., 1989) using SP6 and T7
primers.
8. Thyroglobulin RT-PCR Sensitivity Assay
Thyroglobulin RT-PCR sensitivity was determined by adding thyroid
follicular carcinoma cells (UCLA R0 82 W-1) (Van Herle, A. J., et al.,
1990) into whole blood samples from normal donors. Ten fold serial
dilutions of thyroid follicular carcinoma cells were mixed with 5 mL
aliquots of whole blood and RNA was isolated as described before. RT-PCR
was performed using 1 ug of total RNA and analyzed as described above. In
a second experiment, thyroid follicular carcinoma cells were stimulated in
culture with thyroid stimulating hormone (TSH from bovine pituitary, Sigma
Chem. Co., Missouri) at a concentration of 5 uU/mL for 5 days, and the
stimulated cells were added as above. RNA was isolated and RT-PCR was
performed as before.
RESULTS
1. Detection of Thyroglobulin Transcripts by RT-PCR
FIGS. 1A and 1B show thyroglobulin transcript primers P1 (sense) and P2
(antisense), and their relative positions in the thyroglobulin gene.
Thyroglobulin transcripts were detected as a 529 bp amplicon in the human
thyroid follicular cell line, human thyroid tissue and whole blood from
metastatic thyroid cancer patients by reverse transcription of RNA primers
P1 and P2.
FIG. 6A shows an example of thyroglobulin fragment amplification in
peripheral blood of a patient with benign thyroid disease immediately
after thyroid operation, but no such amplification was evident
preoperatively. Integrity of RNA in all cases was confirmed by running an
RT-PCR in parallel with human glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) primers. In each case, an amplification of 711 bp was evident in
the gel (see FIG. 6B).
2. Restriction Enzyme Fragmentation and Nucleotide Sequence Verification of
RT-PCR Amplified Fragments
All thyroglobulin RT-PCR fragments were further analyzed by Bgl II
restriction enzyme digestion. FIG. 3 shows a few such analyses. As
expected in each case two restriction fragments of 272 and 257 bp were
revealed confirming the restriction site in the amplified thyroglobulin
fragment. Undigested controls were included in each case in the gel
analysis.
In two representative cases the RT-PCR amplified thyroglobulin fragments
were cloned into pCR II vector (Invitrogen, California) and sequenced in
both directions using SP6 and T7 primers by dideoxynucleotide chain
termination method of Sanger et al (Sanger, F., et al., 1977). The derived
nucleotide sequences of RT-PCR amplified thyroglobulin fragments were
identical to the nucleotide sequence of thyroglobulin reported in the Gen
Bank (Accession number 02154) (Jiang, H., et al., 1994) (FIG. 4). This
further confirmed that the amplified fragments were derived from
thyroglobulin transcripts.
3. Patients
Postoperative peripheral blood samples were obtained from 36 patients
undergoing thyroid or parathyroid operation. These patients included 9
with differentiated thyroid cancer, 19 with benign thyroid disease, 3 with
parathyroid adenomas, 4 with differentiated thyroid cancer and parathyroid
adenomas and one with both benign thyroid disease and parathyroid adenoma.
Preoperatively, thyroglobulin transcripts were detected in 10 or 36,
including 2 patients with differentiated thyroid cancer, 6 with benign
thyroid cancer and 2 with parathyroid adenoma. Fourteen of 36 patients
were RT-PCR positive immediately following the operation (5 patients with
differentiated thyroid cancer, 6 with benign thyroid cancer, 2 with
parathyroid adenoma, and 1 with differentiated thyroid cancer and
parathyroid adenoma. Additionally, 10 of the 36 patients had blood samples
drawn three postoperatively. Only 1 of these 10 RT-PCR assays was positive
in a patient with papillary thyroid cancer. These data are displayed in
Table 2.
TABLE 2
______________________________________
Comparison of PCR to Clinical Staging in Immediate Preoperative,
Immediate Postoperative and 3 Week Postoperative Patients
Postoperative
Patient # Diagnosis Preoperative Postoperative (3 wks)
______________________________________
1 C + + NA
2 C - + NA
3 PA - - NA
4 B - - NA
5 B - - NA
6 B - - NA
7 B + + NA
8 PA + + NA
9 C + PA - - -
10 C - + +
11 B - - -
12 PA + + -
13 C - - -
14 C - + -
15 C + PA - - -
16 B + PA - - NA
17 C - - -
18 B - - -
19 B - - NA
20 C + - NA
21 B + + -
22 B + + NA
23 C - - NA
24 C + PA - + NA
25 C + PA - - NA
26 B + - NA
27 B - - NA
28 C - + NA
29 B - + NA
30 B - + NA
31 B - - NA
32 B + - NA
33 B - + NA
34 B + - NA
35 B - - NA
36 B - - NA
______________________________________
B; benign thyroid disease;
C: differentiated thyroid cancer,
PA: parathyroid adenoma,
B + PA: benign thyroid disease and parathyroid adenoma,
C + PA: differentiated thyroid cancer and parathyroid adenoma.
CONCLUSIONS
It is generally recognized that papillary thyroid cancers metastasize to
the lymph nodes, and follicular cancers are hematogenously disseminated.
The findings further support the hypothesis that papillary cancers may
also metastsize hematogenously.
The initial discovery of circulating thyrocytes in the peripheral blood of
postoperative patients with metastatic thyroid cancer led to
investigations into the biological significance of these finds.
Specifically, in patients with intact but diseased thyroids, both benign
and malignant, would these patients shed thyrocytes into the peripheral
circulation? It was hypothesized that both benign and malignant thyroid
disorders might cause these cells to be shed into the bloodstream. The
immediate postoperative period in thyroid cells is a special situation;
manipulation and incising thyroid tissue may allow thyrocytes to enter the
bloodstream and circulate briefly. Therefore, circulating thyroid cells
should be detected in a larger number of patients. However, if there is no
residual disease, the "false" positives should clear several weeks after
surgery. Only those patients with malignant disease capable of
metastasizing through the blood stream should have detectable thyrocytes
which persist after the immediate postoperative period.
Circulating thyrocytes in 28% of the patients who were about to undergo
thyroid or parathyroid surgery were identified. Immediately,
postoperatively, however that number increased to 39%; this increase is
believed due to spillage of thyroid cells during thyroid operation or neck
exploration for hyperparathyroidism. Finally, by three weeks
postoperative, only one of ten patients had residual circulating thyroid
cells. This patient had undergone total thyroidectomy for papillary
thyroid cancer. Thus, it is believed this patient represents a true
positive.
The RT-PCR assay for thyroglobulin transcripts that has been developed
appears to have preoperative limitations. The presence of thyroid disease,
either malignant or benign, may cause a positive assay in the preoperative
period. Additionally, operative thyroid manipulation appears to cause an
increase number of false positives immediately after the operation. By
several week postoperatively, however, the majority of patients clear
these circulating thyrocytes, and evidence of disease. These data support
use of the RT-PCR assay for circulating cells with thyroglobulin
transcripts as long term postoperative tests to identify early metastatic
thyroid disease.
THIRD SET OF EXPERIMENTS
Since thyroid peroxidase is actively expressed only in thyrocytes,
similarly to thyroglobulin, one could utilize the methods described in the
two previously described set of experiments and use RT-PCR to detect mRNA
transcripts of thyroid peroxidase.
Based on the known cDNA sequence of thyroid peroxidase, primers were
custom-designed as described in the earlier experiments. The sequences of
these primers are given in FIGS. 7A and 7B. After amplification, the
RT-PCR fragments were sequenced (FIGS. 7C and 7D).
In combination with the information available, one skilled in the art could
detect thyroid cancer by detecting circulating thyrocytes that actively
express thyroid peroxidase using RT-PCR.
REFERENCES
1. Braverman, L. E., et al. (1991) The thyroid a fundamental and clinical
text. 6th ed. Philadelphia: JB Lippincott Company, p. 1142.
2. Burchill, S. A., et al. (1994) "Neuroblastoma cell detection by reverse
transcriptase-polymerase chain reaction (RT-PCR) for tyrosine hydroxylase
mRNA." Int. J. Cancer 57: 671-675.
3. Cady, B. and Rossi, R. L. (1991) Surgery of the thyroid and parathyroid
glands. 3rd ed. Philadelphia: WB Saunders Company, p. 139.
4. Goldblatt, S. A. and Nadel, E. M. (1965) "Cancer cells in the
circulating blood: a critical review II." Acta Cytologica 9: 6-20.
5. Jiang, H., et al. (1994) "Induction of differentiation in human
promyelocytic HL-60 leukemia cells activates p21, WAF1/CIP1, expression in
the abscence of p53." Oncogene 9: 3397-3406.
6. Johnson, P. W. M., et al. (1995) "The molecular detection of circulating
tumour cells." British Journal of Cancer 72: 268-276.
7. Katz, A. E., et al. (1994) "Molecular staging of prostate cancer with
the use of an enhanced reverse transcriptase-PCR assay." Urology 43:
765-775.
8. Lo Gerfo, P., et al. (1979) "Thyroglobulin in benign and malignant
thyroid disease." JAMA 241: 923-925.
9. Mattano, L. A., et al. (1992) "Sensitive detection of rare circulating
neuroblastoma cells by the reverse transcriptase-polymerase chain
reaction." Cancer Research 52: 4701-4705.
10. Moreno, J. G., et al. (1992) "Detection of hematogenous micrometastasis
in patients with prostate cancer." Cancer Research 52: 6110-6112.
11. Sambrook, J., et al. (1989) Molecular cloning: a laboratory manual. 2nd
ed. New York: Cold Spring Harbor Laboratory Press.
12. Sanger, F., et al. (1977) "DNA sequencing with chain-terminating
inhibitors." Proc. Natl. Acad. Sci. 74: 5463.
13. Seiden, M. V., et al. (1994) "Detection of circulating tumor cells in
men with localized prostate cancer." Journal of Clinical Oncology 12:
2634-2639.
14. Shah, J. P. and Lydiatt, W. (1995) "Treatment of cancer of the head and
neck." CA Cancer J Clin 45: 352-368.
15. Van Herle, A. J., et al. (1990) "Effects of 13 cis-Retinoic acid on
growth and differentiation of human follicular carcinoma cells (UCLA R0 82
W-1) in Vitro." Journal of Clinical Endocrinology and Metabolism 71:
755-763.
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